Skip to main content
Springer Nature Link
Log in

CCL2 promotes P2X4 receptor trafficking to the cell surface of microglia

  • Original Article
  • Published:
Purinergic Signalling Aims and scope Submit manuscript

Abstract

P2X4 receptors (P2X4Rs), a subtype of the purinergic P2X family, play important roles in regulating neuronal and glial functions in the nervous system. We have previously shown that the expression of P2X4Rs is upregulated in activated microglia after peripheral nerve injury and that activation of the receptors by extracellular ATP is crucial for maintaining nerve injury-induced pain hypersensitivity. However, the regulation of P2X4R expression on the cell surface of microglia is poorly understood. Here, we identify the CC chemokine receptor CCR2 as a regulator of P2X4R trafficking to the cell surface of microglia. In a quantitative cell surface biotinylation assay, we found that applying CCL2 or CCL12, endogenous ligands for CCR2, to primary cultured microglial cells, increased the levels of P2X4R protein on the cell surface without changing total cellular expression. This effect of CCL2 was prevented by an antagonist of CCR2. Time-lapse imaging of green fluorescent protein (GFP)-tagged P2X4R in living microglial cells showed that CCL2 stimulation increased the movement of P2X4R-GFP particles. The subcellular localization of P2X4R immunofluorescence was restricted to lysosomes around the perinuclear region. Notably, CCL2 changed the distribution of lysosomes with P2X4R immunofluorescence within microglial cells and induced release of the lysosomal enzyme β-hexosaminidase, indicating lysosomal exocytosis. Moreover, CCL2-stimulated microglia enhanced Akt phosphorylation by ATP applied extracellularly, a P2X4R-mediated response. These results indicate that CCL2 promotes expression of P2X4R protein on the cell surface of microglia through exocytosis of P2X4R-containing lysosomes, which may be a possible mechanism for pain hypersensitivity after nerve injury.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price includes VAT (Canada)

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  1. Hanisch UK, Kettenmann H (2007) Microglia: active sensor and versatile effector cells in the normal and pathologic brain. Nat Neurosci 10:1387–1394

    Article  PubMed  CAS  Google Scholar 

  2. Streit WJ (2002) Microglia as neuroprotective, immunocompetent cells of the CNS. Glia 40:133–139

    Article  PubMed  Google Scholar 

  3. Prinz M, Mildner A (2011) Microglia in the CNS: immigrants from another world. Glia 59:177–187

    Article  PubMed  Google Scholar 

  4. Inoue K, Koizumi S, Tsuda M (2007) The role of nucleotides in the neuron–glia communication responsible for the brain functions. J Neurochem 102:1447–1458

    Article  PubMed  CAS  Google Scholar 

  5. Pocock JM, Kettenmann H (2007) Neurotransmitter receptors on microglia. Trends Neurosci 30:527–535

    Article  PubMed  CAS  Google Scholar 

  6. Tsuda M, Shigemoto-Mogami Y, Koizumi S et al (2003) P2X4 receptors induced in spinal microglia gate tactile allodynia after nerve injury. Nature 424:778–783

    Article  PubMed  CAS  Google Scholar 

  7. Cavaliere F, Florenzano F, Amadio S et al (2003) Up-regulation of P2X2, P2X4 receptor and ischemic cell death: prevention by P2 antagonists. Neuroscience 120:85–98

    Article  PubMed  CAS  Google Scholar 

  8. Guo LH, Trautmann K, Schluesener HJ (2004) Expression of P2X4 receptor in rat C6 glioma by tumor-associated macrophages and activated microglia. J Neuroimmunol 152:67–72

    Article  PubMed  CAS  Google Scholar 

  9. Guo LH, Trautmann K, Schluesener HJ (2005) Expression of P2X4 receptor by lesional activated microglia during formalin-induced inflammatory pain. J Neuroimmunol 163:120–127

    Article  PubMed  CAS  Google Scholar 

  10. Zhang Z, Zhang Z, Artelt M et al (2007) Dexamethasone attenuates early expression of three molecules associated with microglia/macrophages activation following rat traumatic brain injury. Acta Neuropathol 113:675–682

    Article  PubMed  CAS  Google Scholar 

  11. Schwab JM, Guo L, Schluesener HJ (2005) Spinal cord injury induces early and persistent lesional P2X4 receptor expression. J Neuroimmunol 163:185–189

    Article  PubMed  CAS  Google Scholar 

  12. Zhang Z, Zhang ZY, Fauser U et al (2008) Mechanical allodynia and spinal up-regulation of P2X4 receptor in experimental autoimmune neuritis rats. Neuroscience 152:495–501

    Article  PubMed  CAS  Google Scholar 

  13. Coull JA, Beggs S, Boudreau D et al (2005) BDNF from microglia causes the shift in neuronal anion gradient underlying neuropathic pain. Nature 438:1017–1021

    Article  PubMed  CAS  Google Scholar 

  14. Tsuda M, Inoue K, Salter MW (2005) Neuropathic pain and spinal microglia: a big problem from molecules in "small" glia. Trends Neurosci 28:101–107

    Article  PubMed  CAS  Google Scholar 

  15. Ohsawa K, Irino Y, Nakamura Y et al (2007) Involvement of P2X4 and P2Y12 receptors in ATP-induced microglial chemotaxis. Glia 55:604–616

    Article  PubMed  Google Scholar 

  16. Ohsawa K, Irino Y, Sanagi T et al (2010) P2Y12 receptor-mediated integrin-beta1 activation regulates microglial process extension induced by ATP. Glia 58:790–801

    PubMed  Google Scholar 

  17. Wu LJ, Vadakkan KI, Zhuo M (2007) ATP-induced chemotaxis of microglial processes requires P2Y receptor-activated initiation of outward potassium currents. Glia 55:810–821

    Article  PubMed  Google Scholar 

  18. Charo IF, Myers SJ, Herman A et al (1994) Molecular cloning and functional expression of two monocyte chemoattractant protein 1 receptors reveals alternative splicing of the carboxyl-terminal tails. Proc Natl Acad Sci U S A 91:2752–2756

    Article  PubMed  CAS  Google Scholar 

  19. Boumechache M, Masin M, Edwardson JM et al (2009) Analysis of assembly and trafficking of native P2X4 and P2X7 receptor complexes in rodent immune cells. J Biol Chem 284:13446–13454

    Article  PubMed  CAS  Google Scholar 

  20. Tsuda M, Masuda T, Kitano J et al (2009) IFN-gamma receptor signaling mediates spinal microglia activation driving neuropathic pain. Proc Natl Acad Sci U S A 106:8032–8037

    Article  PubMed  CAS  Google Scholar 

  21. Nasu-Tada K, Koizumi S, Tsuda M et al (2006) Possible involvement of increase in spinal fibronectin following peripheral nerve injury in upregulation of microglial P2X4, a key molecule for mechanical allodynia. Glia 53:769–775

    Article  PubMed  Google Scholar 

  22. Tsuda M, Toyomitsu E, Kometani M et al (2009) Mechanisms underlying fibronectin-induced up-regulation of P2X4R expression in microglia: distinct roles of PI3K-Akt and MEK-ERK signalling pathways. J Cell Mol Med 13:3251–3259

    Article  PubMed  Google Scholar 

  23. Tsuda M, Toyomitsu E, Komatsu T et al (2008) Fibronectin/integrin system is involved in P2X(4) receptor upregulation in the spinal cord and neuropathic pain after nerve injury. Glia 56:579–585

    Article  PubMed  Google Scholar 

  24. Tsuda M, Tozaki-Saitoh H, Masuda T et al (2008) Lyn tyrosine kinase is required for P2X(4) receptor upregulation and neuropathic pain after peripheral nerve injury. Glia 56:50–58

    Article  PubMed  Google Scholar 

  25. Qureshi OS, Paramasivam A, Yu JC et al (2007) Regulation of P2X4 receptors by lysosomal targeting, glycan protection and exocytosis. J Cell Sci 120:3838–3849

    Article  PubMed  CAS  Google Scholar 

  26. Schwartz M, Butovsky O, Bruck W et al (2006) Microglial phenotype: is the commitment reversible? Trends Neurosci 29:68–74

    Article  PubMed  CAS  Google Scholar 

  27. Nakajima K, Shimojo M, Hamanoue M et al (1992) Identification of elastase as a secretory protease from cultured rat microglia. J Neurochem 58:1401–1408

    Article  PubMed  CAS  Google Scholar 

  28. Kuronita T, Eskelinen EL, Fujita H et al (2002) A role for the lysosomal membrane protein LGP85 in the biogenesis and maintenance of endosomal and lysosomal morphology. J Cell Sci 115:4117–4131

    Article  PubMed  CAS  Google Scholar 

  29. Mirzadegan T, Diehl F, Ebi B et al (2000) Identification of the binding site for a novel class of CCR2b chemokine receptor antagonists: binding to a common chemokine receptor motif within the helical bundle. J Biol Chem 275:25562–25571

    Article  PubMed  CAS  Google Scholar 

  30. Nanki T, Nagasaka K, Hayashida K et al (2001) Chemokines regulate IL-6 and IL-8 production by fibroblast-like synoviocytes from patients with rheumatoid arthritis. J Immunol 167:5381–5385

    PubMed  CAS  Google Scholar 

  31. Jimenez-Sainz MC, Fast B, Mayor F Jr et al (2003) Signaling pathways for monocyte chemoattractant protein 1-mediated extracellular signal-regulated kinase activation. Mol Pharmacol 64:773–782

    Article  PubMed  CAS  Google Scholar 

  32. Myers SJ, Wong LM, Charo IF (1995) Signal transduction and ligand specificity of the human monocyte chemoattractant protein-1 receptor in transfected embryonic kidney cells. J Biol Chem 270:5786–5792

    Article  PubMed  CAS  Google Scholar 

  33. Wong LM, Myers SJ, Tsou CL et al (1997) Organization and differential expression of the human monocyte chemoattractant protein 1 receptor gene. Evidence for the role of the carboxyl-terminal tail in receptor trafficking. J Biol Chem 272:1038–1045

    Article  PubMed  CAS  Google Scholar 

  34. Sarafi MN, Garcia-Zepeda EA, MacLean JA et al (1997) Murine monocyte chemoattractant protein (MCP)-5: a novel CC chemokine that is a structural and functional homologue of human MCP-1. J Exp Med 185:99–109

    Article  PubMed  CAS  Google Scholar 

  35. Inoue K, Tsuda M (2009) Microglia and neuropathic pain. Glia 57:1469–1479

    Article  PubMed  Google Scholar 

  36. Royle SJ, Qureshi OS, Bobanovic LK et al (2005) Non-canonical YXXGPhi endocytic motifs: recognition by AP2 and preferential utilization in P2X4 receptors. J Cell Sci 118:3073–3080

    Article  PubMed  CAS  Google Scholar 

  37. Bobanovic LK, Royle SJ, Murrell-Lagnado RD (2002) P2X receptor trafficking in neurons is subunit specific. J Neurosci 22:4814–4824

    PubMed  CAS  Google Scholar 

  38. Eskelinen EL, Tanaka Y, Saftig P (2003) At the acidic edge: emerging functions for lysosomal membrane proteins. Trends Cell Biol 13:137–145

    Article  PubMed  CAS  Google Scholar 

  39. Ulmann L, Hatcher JP, Hughes JP et al (2008) Up-regulation of P2X4 receptors in spinal microglia after peripheral nerve injury mediates BDNF release and neuropathic pain. J Neurosci 28:11263–11268

    Article  PubMed  CAS  Google Scholar 

  40. Burnstock G (2008) Purinergic signalling and disorders of the central nervous system. Nat Rev Drug Discov 7:575–590

    Article  PubMed  CAS  Google Scholar 

  41. Stokes L, Surprenant A (2009) Dynamic regulation of the P2X4 receptor in alveolar macrophages by phagocytosis and classical activation. Eur J Immunol 39:986–995

    Article  PubMed  CAS  Google Scholar 

  42. Toulme E, Garcia A, Samways D et al (2010) P2X4 receptors in activated C8-B4 cells of cerebellar microglial origin. J Gen Physiol 135:333–353

    Article  PubMed  CAS  Google Scholar 

  43. Van Steenwinckel J, Reaux-Le Goazigo A, Pommier B et al (2011) CCL2 released from neuronal synaptic vesicles in the spinal cord is a major mediator of local inflammation and pain after peripheral nerve injury. J Neurosci 31:5865–5875

    Article  PubMed  Google Scholar 

  44. Murrell-Lagnado RD, Qureshi OS (2008) Assembly and trafficking of P2X purinergic receptors (review). Mol Membr Biol 25:321–331

    Article  PubMed  CAS  Google Scholar 

  45. Colvin RA, Means TK, Diefenbach TJ et al (2010) Synaptotagmin-mediated vesicle fusion regulates cell migration. Nat Immunol 11:495–502

    Article  PubMed  CAS  Google Scholar 

  46. Czibener C, Sherer NM, Becker SM et al (2006) Ca2+ and synaptotagmin VII-dependent delivery of lysosomal membrane to nascent phagosomes. J Cell Biol 174:997–1007

    Article  PubMed  CAS  Google Scholar 

  47. Wu D, LaRosa GJ, Simon MI (1993) G protein-coupled signal transduction pathways for interleukin-8. Science 261:101–103

    Article  PubMed  CAS  Google Scholar 

  48. Kuehn HS, Gilfillan AM (2007) G protein-coupled receptors and the modification of FcepsilonRI-mediated mast cell activation. Immunol Lett 113:59–69

    Article  PubMed  CAS  Google Scholar 

  49. Serrano A, Pare M, McIntosh F et al (2010) Blocking spinal CCR2 with AZ889 reversed hyperalgesia in a model of neuropathic pain. Mol Pain 6:90

    Article  PubMed  CAS  Google Scholar 

  50. Old EA, Malcangio M (2011) Chemokine mediated neuron-glia communication and aberrant signalling in neuropathic pain states. Curr Opin Pharmacol (in press)

  51. Terashima Y, Onai N, Murai M et al (2005) Pivotal function for cytoplasmic protein FROUNT in CCR2-mediated monocyte chemotaxis. Nat Immunol 6:827–835

    Article  PubMed  CAS  Google Scholar 

  52. Schilling M, Strecker JK, Ringelstein EB et al (2009) The role of CC chemokine receptor 2 on microglia activation and blood-borne cell recruitment after transient focal cerebral ischemia in mice. Brain Res 1289:79–84

    Article  PubMed  CAS  Google Scholar 

  53. Galasso JM, Stegman LD, Blaivas M et al (2000) Experimental gliosarcoma induces chemokine receptor expression in rat brain. Exp Neurol 161:85–95

    Article  PubMed  CAS  Google Scholar 

  54. Abbadie C, Bhangoo S, De Koninck Y et al (2009) Chemokines and pain mechanisms. Brain Res Rev 60:125–134

    Article  PubMed  CAS  Google Scholar 

  55. Abbadie C, Lindia JA, Cumiskey AM et al (2003) Impaired neuropathic pain responses in mice lacking the chemokine receptor CCR2. Proc Natl Acad Sci U S A 100:7947–7952

    Article  PubMed  CAS  Google Scholar 

  56. Wixey JA, Reinebrant HE, Carty ML et al (2009) Delayed P2X4R expression after hypoxia-ischemia is associated with microglia in the immature rat brain. J Neuroimmunol 212:35–43

    Article  PubMed  CAS  Google Scholar 

  57. Zhang J, Shi XQ, Echeverry S et al (2007) Expression of CCR2 in both resident and bone marrow-derived microglia plays a critical role in neuropathic pain. J Neurosci 27:12396–12406

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (M.T., K.I.) and from the Core-to-Core program “Integrated Action Initiatives” of the Japan Society for the Promotion of Science (JSPS) (M.T., K.I.).

Author information

Authors and Affiliations

  1. Department of Molecular and System Pharmacology, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan

    Emika Toyomitsu, Makoto Tsuda, Tomohiro Yamashita, Hidetoshi Tozaki-Saitoh & Kazuhide Inoue

  2. Department of Pharmaceutical Cell Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, 812-8582, Japan

    Yoshitaka Tanaka

Authors
  1. Emika Toyomitsu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Makoto Tsuda
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Tomohiro Yamashita
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hidetoshi Tozaki-Saitoh
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yoshitaka Tanaka
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Kazuhide Inoue
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Kazuhide Inoue.

Additional information

E. Toyomitsu and M. Tsuda are equally contributed to this work.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Toyomitsu, E., Tsuda, M., Yamashita, T. et al. CCL2 promotes P2X4 receptor trafficking to the cell surface of microglia. Purinergic Signalling 8, 301–310 (2012). https://doi.org/10.1007/s11302-011-9288-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11302-011-9288-x

Keywords

Search

Navigation